Mating in the budding yeast S. cerevisiae provides an excellent paradigm for negative growth control and differentiation in higher eukaryotes. In response to peptide pheromones, dividing haploid cells stop dividing in G1 phase and express specialized genes required for cell-cell contact and fusion. Mating is regulated by a conserved MAP kinase cascade that consists of two MAP kinases, a MEK, a MEKK, and a PAK-type kinase. The cascade is regulated by heterotrimeric and Rho-type G proteins, an SH3 domain protein, and a novel scaffolding protein called Ste5 that spatially organizes the protein kinases. This proposal aims to study two fundamental problems of signal transduction that pertain to growth and differentiation in all eukaryotes--(1) the mechanism by which a MAP kinase cascade is activated and (2) how a MAP kinase cascade performs specialized functions. The grant consists of distinct and interrelated projects, all of which are a continuation of work done during the previous granting period. The PI proposes to: 1. Investigate the different possible functions of Ste5 with respect to protein kinase regulation by isolating and analyzing Ste5 mutants defective in specific functions and interactions with protein kinases and G proteins. Mutant proteins will be characterized biochemically for the ability to bind to pathway components, activate kinases, and direct substrate phosphorylation. 2. Cell biological and genetic approaches will be used to investigate the role of cell cycle- and pheromone-dependent regulation of Ste5 localization to determine whether it is important for sequestering MAPK cascade enzymes to different cellular compartments for specific responses. 3. Mechanisms of activation of the MEKK Ste11 will be analyzed with respect to both Ste11 and Ste5 dimerization and association between the regulatory domain of Ste11 and Ste5 and Ste50. 4. The role of Ste5 in mediating pathway specificity will be investigated by isolating and analyzing mutant forms of Ste5 and Ste11 that activate other MAP kinase cascades.